selleck inhibitor Phosphorylation of ERK1/2 and NFκB activation are primarily responsible for protecting ILK KO hepatocytes from apoptosis Consistent with our in vivo data, hepatocytes isolated from ILK KO mice were resistant to Jo-2 and Actinomycin D induced apoptosis (Figure 3A). Our in vivo data suggest that increase in survival pathways like Akt, Erk1/2 and NFκB

plays a role in affording this buy Sapanisertib protection. We used pharmacological inhibitors for Akt and Erk1/2 and peptide inhibitor for NFκB. Inhibition of Erk1/2 and NFκB led to increased susceptibility of ILK KO hepatocytes to Jo-2 and Actinomycin D induced apoptosis (Figure 3B and 3C). Pharmacological inhibitor against ERK1/2 was effective in downregulating the phosphorylation of ERK (Figure 3D). Inhibition of Akt did not have any effect (Figure 3B). Thus, NFκB and Erk1/2 but not Akt seem to be involved

in affording protection to ILK KO hepatocytes to Jo-2 and actinomycin D induced apoptosis. Figure 3 ILK KO hepatocytes are protected against Jo-2 induced apoptosis in vitro. A) Caspase 3/7 activity at 6 h after treatment of WT and ILK KO hepatocytes with Jo-2 (0.5 μg/ml) and Actinomycin D (0.05 μg/ml). Fold change is the ratio of luminescence value of treatment group with its corresponding no treatment group. B) Effect of ERK1/2 inhibition using a MEK inhibitor U0126 (20 μM). Representative Western blots of cleaved caspase and PARP 6 h after Jo-2, vehicle or Jo-2+inhibitor administration. (Akt Inh: Akt inhibitor LY-294002, ERK Inh: ERK inhibitor U0126) C) Representative Western blots showing inhibition of phosphorylation SNX-5422 of ERK1/2 by U0126 in ILK KO hepatocytes after 6 h after treatment with U0126. D) Representative Western blots of PARP after inhibition of NFκB using a synthetic peptide 6 h after treatment with Control peptide (CP), CP+Jo-2 and NP+Jo-2 (NFκB peptide). (CP: control peptide, NP: NFκB peptide). E) Total FAK and p-FAK at 0 and 6 h after Jo-2 C59 mw administration in vitro.

F) Total FAK and p-FAK at 0 and 6 h after Jo-2 administration in vivo. Focal adhesion kinase signaling Focal adhesion kinase is another enzyme associated with integrin signaling [18, 19]. We looked into the possibility of FAK signaling compensating for the loss of ILK signaling. Genetic removal of ILK led to lower expression of FAK in the whole liver as well as hepatocytes isolated from the ILK KO mice (0 h of Figure 3E and 3F). Activation of FAK as a result of tyrosine phosphorylation at 397 residue was also lower in the whole liver as well as hepatocytes of the ILK KO mice (0 h of Figure 3E and 3F). Interestingly, administration of Jo-2 both in vivo and in vitro resulted in an increase in total as well as activated FAK in the ILK KO mice (Figure 3E and 3F). The WT mice on the other hand showed downregulation of total and activated FAK after Jo-2 administration both in vivo and in vitro.